Similar and divergent responses to salinity stress of jamun (Syzygium cumini L. Skeels) genotypes

Background Genetic variation for salt tolerance remains elusive in jamun (Syzygium cumini). Methods Effects of gradually increased salinity (2.0–12.0 dS/m) were examined in 20 monoembryonic and 28 polyembryonic genotypes of jamun. Six genotypes were additionally assessed for understanding salt-induced changes in gas exchange attributes and antioxidant enzymes. Results Salt-induced reductions in leaf, stem, root and plant dry mass (PDM) were relatively greater in mono- than in poly-embryonic types. Reductions in PDM relative to control implied more adverse impacts of salinity on genotypes CSJ-28, CSJ-31, CSJ-43 and CSJ-47 (mono) and CSJ-1, CSJ-24, CSJ-26 and CSJ-27 (poly). Comparably, some mono- (CSJ-5, CSJ-18) and poly-embryonic (CSJ-7, CSJ-8, CSJ-14, CSJ-19) genotypes exhibited least reductions in PDM following salt treatment. Most polyembryonic genotypes showed lower reductions in root than in shoot mass, indicating that they may be more adept at absorbing water and nutrients when exposed to salt. The majority of genotypes did not exhibit leaf tip burn and marginal scorch despite significant increases in Na+ and Cl−, suggesting that tissue tolerance existed for storing excess Na+ and Cl− in vacuoles. Jamun genotypes were likely more efficient in Cl− exclusion because leaf, stem and root Cl− levels were consistently lower than those of Na+ under salt treatment. Leaf K+ was particularly little affected in genotypes with high leaf Na+. Lack of discernible differences in leaf, stem and root Ca2+ and Mg2+ contents between control and salt treatments was likely due to their preferential uptake. Correlation analysis suggested that Na+ probably had a greater inhibitory effect on biomass in both mono- and poly-embryonic types. Discriminant analysis revealed that while stem and root Cl− probably accounted for shared responses, root Na+, leaf K+ and leaf Cl− explained divergent responses to salt stress of mono- and poly-embryonic types. Genotypes CSJ-18 and CSJ-19 seemed efficient in fending off oxidative damage caused by salt because of their stronger antioxidant defences. Conclusions Polyembryonic genotypes CSJ-7, CSJ-8, CSJ-14 and CSJ-19, which showed least reductions in biomass even after prolonged exposure to salinity stress, may be used as salt-tolerant rootstocks. The biochemical and molecular underpinnings of tissue tolerance to excess Na+ and Cl− as well as preferential uptake of K+, Ca2+, and Mg2+ need to be elucidated.

chemoprotective properties (Kapoor, Ranote & Sharma, 2015;Shrikanta, Kumar & Govindaswamy, 2015).Genetic variations for salt tolerance in Jamun remain elusive, notwithstanding some anecdotal evidence suggesting that this species flourishes under saline conditions (Sarvade et al., 2016;Tewari, Singh & Nainwal, 2017).This is because the prior salinity experiments in Jamun did not even examine the salt-induced changes in biomass allocation and ion partitioning (Chhabra & Kumar, 2008;Tomar et al., 2003).Jamun exhibits polyembryony, characterized by the emergence of more than one seedling from a single seed (Sivasubramaniam & Selvarani, 2012).Despite the fact that polyembryonic saplings may perform better under salt stress (Nimbolkar et al., 2019), the comparative reactions to salt stress of mono-and poly-embryonic seedlings of jamun are not known.
Considering the previously highlighted gaps in research, this experiment was conducted to assess the effects of incremental rise in salinity on biomass allocation and ion partitioning in 20 monoembryonic and 28 polyembryonic genotypes of jamun with the aim of identifying major traits that underpin salt tolerance.The polyembryonic seedlings were distinguished from the monoembryonic types on the basis growth habit i.e., the presence of multiple seedlings per seed.The shared and contrasting responses to salt of mono-and poly-embryonic seedlings were also examined.Six randomly chosen genotypes were additionally assessed to elucidate salt-induced changes in gas exchange attributes and antioxidant enzymes.

Study site and experimental conditions
The experiment was conducted at ICAR-Central Soil Salinity Research Institute, Karnal,India (29 42′31.7″N 76 57′12.7″E) between December 2018 and June 2019.The study area experiences a subtropical climate, with scorching summers, dry winters and total annual rainfall of about 700 mm.Forty-eight (48) genotypes of jamun, comprising both mono-(20) and poly-embryonic (28) types, were examined (Table 1).Approximately six-monthold seedlings were moved to clay pots (upper diameter: 20 cm, basal diameter: 12 cm, height: 18 cm) having 8 kg soil, river sand and farmyard manure (1: 1: 1 v/v) on December 7, 2019.The drainage hole at the bottom of pots was covered with glass wool to prevent the potting soil from leaking out.The experimental plants were kept in a net-house, open on all sides but covered with a polyethylene sheet to prevent the intrusion of rainwater.The experimental pots were reshuffled every 2 weeks to minimize the spatial effects.

Salinity treatments
Assuming that an incremental rise in salinity would more closely resemble soil salinity under field conditions, where plants rarely encounter sudden surge in salt stress (Shavrukov, 2013), salinity of irrigation water was gradually raised every week, beginning at 2 dS/m and increasing to 12 dS/m over time.Salt treatments were imposed on December 21, 2018, 2 weeks after planting.Saline groundwater (electrical conductivity ~15.0-16.0dS/m) was diluted with fresh water to obtain waters of varying electrical conductivities (2,4,6,8,10 and 12 dS/m).Fresh water (0.70 dS/m) was used to irrigate the control plants.shows the compositions of fresh and saline waters.Irrigation water was poured in using a graduated beaker until it evenly reached the bottom of the pots.The plants were watered until May 31 2019, when treatments were stopped with the appearance of leaf injury symptoms in some mono-(CSJ-28, CSJ-31) and poly-embryonic (CSJ-1, CSJ-34) genotypes.

Observations recorded
Leaf, stem and root biomass The observations were recorded 5 weeks after salinity level of 12.0 dS/m was imposed.The plants were gently uprooted, cleaned on a wire screen, and briefly shade dried.Then, the individual plants were separated into leaves (excluding 4 th pair from apex), stems and roots, washed using distilled water, put within envelopes, and oven-dried to a constant weight (NSW, Gurugram, India).Leaf, stem and root dry mass were recorded using an electronic balance.Plant dry mass was determined by adding the mass of each component.

Gas exchange attributes and anti-oxidant enzymes
The mature leaves (4 th pair from apex) were tagged for recording the photosynthetic attributes and anti-oxidant enzymes.Six genotypes (CSJ-1,  showing noticeably distinct responses to salinity stress were randomly selected to assess the salt-induced changes in gas exchange attributes and anti-oxidant enzymes.Net photosynthesis (P n ), transpiration rate (E) and internal CO 2 concentration (C i ) were measured using a portable photosynthesis system (6400 XT; LI-COR, Lincoln, NE, USA) (Singh et al., 2024).The ratio between P n and E was used to compute the instantaneous water usage efficiency (WUE).Ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities were determined using the procedures described in Nakano & Asada (1981) and Beauchamp & Fridovich (1971), respectively.The methodologies given in Aebi (1984) and Rao et al. (1998) were adopted to measure catalase (CAT) and peroxidase (POX) activities, respectively.

Mineral ions
For ion analyses, finely ground leaf (4 th pair from apex), stem and root tissues (50 mg each) were used.A flame photometer (Systronics, Ahmedabad, India) was used to measure Na + and K + , an ion-selective electrode (Eutech, Singapore) for determining Cl − , and an atomic absorption spectrometer (Analytik Jena, Jena, Germany) for measuring Ca 2+ and Mg 2+ contents.

Statistical analyses
A randomized block design with four replications was used.The independent and interaction effects of salinity (fixed factor) and genotype (random factor) on the variance in different traits were assessed by a two-way analysis of variance (Doncaster & Davey, 2007).The comparative reactions of mono-and poly-embryonic types to fresh and salt water treatments were examined by Welch's t-test (JASP v. 0.17.3).Welch's test is considered to be more appropriate when sample sizes are unequal.The strength and directionality of associations between the measured traits were determined by computing the Pearson's bivariate correlations.Linear discriminant analysis (LDA) was used for discerning the shared and contrasting responses to salinity stress.A confusion matrix was generated to predict the group membership from LDA (PAST v. 4.10).

Root ions
In monoembryonic types, salt-induced increases in root Na + and Cl − were quite similar (~39.0%);however, K + , Ca 2+ and Mg 2+ contents were not significantly affected.

Comparative responses under control and saline conditions
The polyembryonic types showed significantly higher LDM (t = 5.77, p = < 0.001) and PDM (t = 3.30, p = 0.001) than monoembryonic types under control treatment.Similarly, they also exhibited significantly higher (p < 0.001) LDM, RDM and PDM (16.53, 9.41 and 31.26g/plant, respectively) than monoembryonic types (14.35, 7.97 and 27.31 g/plant, respectively) when treated with salt.Shoot: root ratio was significantly higher (p = 0.009) in monoembryonic types under salt treatment (Table S4; Fig. 1).Of the leaf ions, the two groups differed in Na + only in control and in Cl only in salt treatment.Comparably, K + and Ca 2+ were different under both fresh and salt treatments.Leaf Mg + did not differ significantly under both the conditions (Fig. 2A).Of the stem ions, differences between mono-and poly-embryonic types for Na + were significant only in control and for K + and Ca 2+ in salt treatment (Fig. 2B).In roots, only Na + (10.57%) and Ca 2+ (32.50%) were significantly higher in polyembryonic types under salt treatment; the differences were non-significant for other ions under both control and salt treatments (Table S4; Fig. 2C).

Correlation analysis
Table S5 shows the Pearson's correlations between the measured traits.LDM, SDM, RDM and PDM were all significantly positively correlated with each other, irrespective of seedling type.Leaf Na + had highly significant negative correlations with LDM, SDM, RDM and PDM.Although all biomass attributes exhibited significant negative correlations with leaf Cl − , the degree of association was invariably lower than that between biomass traits and leaf Na + .While relationships of leaf K + and stem Na + with biomass attributes were respectively significantly positive and significantly negative.Stem Cl -had significant negative relationships with LDM and PDM in both mono-and poly-embryonic types, and with SDM only in polyembryonic types.Stem K + had significant positive correlations with all the biomass traits in monoembryonic types, but only with LDM and PDM in the polyembryonic types.The negative correlations between root Na + and biomass traits were greater in poly-than in mono-embryonic types.

Linear discriminant analysis
Table S6 and Fig. 3 display the results of LDA.The first two discriminant functions alone explained approximately 98.0% of the cumulative variance in the data, indicating that LDA efficiently reduced the dimensionality.We found that while LD-1 was mainly a construct of stem Cl − and root Cl − , LD-2 had root Na + , leaf K + and leaf Cl − as the highly weighted variables.The differences between mono-and poly-embryonic types for stem and root Cl − contents were non-significant under both control and salinity treatments.Conversely, the differences between two groups were significant for root Na + , leaf K + , and leaf Cl − under salt treatment (Table S6).Therefore, we infer that while stem and root Cl -contents accounted for shared responses, root Na + , leaf K + and leaf Cl − explained the divergent responses to salinity stress.A perusal of the LDA biplot showed that while LD1 effectively discriminated the control and salinity treatments, LD2 could fairly reasonably distinguish monoembryonic and polyembryonic types from each other (Fig. 3).The confusion matrix estimates for predicted group membership from LDA are presented in Table S7.
The overall classification accuracy (jacknifed) of LDA was 80.21%.In the case of monoembryonic types, 25.0% and 18.75% of the instances were mislabelled as polyembryonic in the control and salt treatments, respectively.Similarly, 19.64% and 13.39% of the polyembryonic instances were incorrectly classified as monoembryonic under control and salinity treatments, respectively.Interestingly, a small proportion of the polyembryonic instances (3.57%) were also incorrectly labelled as monoembryonic in the salt treatment.

DISCUSSION
Little is known about traits underlying salinity tolerance, as well as genetic variations for salt tolerance, in Jamun.In this backdrop, our study aimed to elucidate the effects of salinity stress on biomass allocation and ion uptake in 48 diverse genotypes of jamun.Similar and contrasting responses to salt stress of mono-and poly-embryonic seedlings were also analyzed to identify the mechanisms underlying salinity tolerance.In order to avoid salt shock, irrigation water salinity was increased gradually (Singh et al., 2024).Since 'osmotic' rather than 'salinity' stress usually triggers early reactions under saline conditions, long-term studies are probably more dependable for analyzing salt tolerance (Zhu et al., 2016).By using this method, we were also able in better mimicking the field conditions where salinity typically peaks in the summer (Sadder et al., 2021).The genetic variability for salt tolerance remains elusive in Jamun because prior studies had examined the effects of salt stress only on one genotype (Chhabra & Kumar, 2008;Patil & Patil, 1983;Tomar et al., 2003).Crop genotypes differ markedly in salt tolerance (Liu et al., 2020;Mousavi et al., 2019), suggesting that a sizeable number of genotypes must be evaluated to reasonably assess the variability for salt tolerance.We noticed substantial genotypic differences (p < 0.001) for salt-induced declines in the dry mass of different plant parts.
In monoembryonic types, while CSJ-28 was most adversely affected, genotypes CSJ-5, CSJ-18 and CSJ-38 exhibited the lowest drops in leaf, stem and root biomass.Contrasting genetic variation was also observed within polyembryonic types for the reductions caused by salt in LDM, SDM and RDM.Salt stress suppresses the leaf area, damages the cell membranes, impairs the water relations, causes oxidative stress and hampers the photosynthetic assimilation, which then adversely impact plant growth (Gholami et al., 2023;Moula et al., 2020).The repressive effects of salinity on shoot and root growth vary with genotype in a particular fruit crop (Liu et al., 2020;Moula et al., 2020).We found that while several monoembryonic genotypes (e.g., CSJ-2, CSJ-18 and CSJ-28) showed comparable reductions, a few (e.g., CSJ-5, CSJ-12 and CSJ-45) showed higher decreases in root mass, and the remaining showed greater declines in shoot mass.Likewise, salt-induced reductions in root mass were either lower or substantially lower than superoxide dismutase (all anti-oxidants are in units/g FW).S, salinity; G, genotype; * significant at p < 0.05; ** significant at p < 0.01; *** significant at p < 0.001; ns, non-significant.C and S denote control (0.70 dS/m) and salinity (12.0 dS/m) treatments, respectively.Each value represents mean ± SD.Differences between means that share a letter within each column are not statistically significant (p 0.05).
When treated with salt, the polyembryonic types had significantly higher LDM, RDM and PDM than monoembryonic types.While the differences in leaf and stem Na + were not statistically significant, polyembryonic types retained significantly more Na + in their roots under salinity stress.Comparably, leaf Cl − was noticeably lower in salt-stressed monoembryonic types.This suggested comparatively greater capacity for Cl − exclusion in monoembryonic seedlings, and for Na + exclusion in polyembryonic types (Dayal et al., 2014;Hussain et al., 2012).This is probably because different mechanisms regulate the absorption and partitioning of Na + and Cl − in salt-stressed plants (Saleh et al., 2008).Importantly, polyembryonic types maintained significantly higher levels of leaf K + , leaf Ca 2+ , stem K + , stem Ca 2+ , and root Ca 2+ when exposed to salt.In addition to boosting osmotic adjustment (Mahouachi, 2018) and improving cell membrane stability (Cimato et al., 2010), this may have also restricted Na + uptake (Gengmao et al., 2015), leading to higher leaf and root biomass in the polyembryonic types.Our results broadly concur with earlier findings in mango (Pandey et al., 2014) and citrus (Hussain et al., 2012), which suggest that polyembryonic genotypes may perform better under salt stress.
Correlation analysis indicated that Na + probably had a stronger restrictive effect on leaf, stem and root biomass than Cl − , regardless of the seedling type.The greater inhibitory effects of Na + on plant growth are known in citrus (Balal et al., 2012) and olive (Perica, Goreta & Selak, 2008).In our study, salt stress caused increased build-up of Na + in different plant parts.Na + transport in plants is primarily unidirectional with little recirculation from shoots to roots, which causes Na + to gradually build-up in shoots.The higher Na + levels then cause metabolic toxicity by competing with K + in cellular functions (Tester & Davenport, 2003).Contrarily, phloem recirculation seems to limit Cl − accumulation in aerial plant parts (Godfrey et al., 2019).Thus, we suppose that phloem recirculation might shield jamun plants against Cl − toxicity ( Brumos et al., 2010).Significant positive correlations between biomass attributes and leaf K + implied that enhanced accumulation of K + may contribute to osmotic adjustment (Pérez-Pérez et al., 2009) and facilitate sequestration of excess Na + into vacuoles (Zarei et al., 2016).
Approximately 98.0% of the cumulative variance in the data was described by the first two linear discriminant functions alone, demonstrating the robustness of LDA in reducing the dimensionality (Ye & Ji, 2010).Because the variables (stem and root Cl -) loaded heavily on first linear discriminant function (LD-1) were not significantly different between both the groups under control and saline conditions, we assume that this discriminant function represented the shared responses to salinity stress.Similarly, we assume that LD-2 represented the divergent responses to salinity stress, since the major variables on LD-2 (root Na + , leaf K + and leaf Cl − ) significantly differed between the two groups under salt treatment.Earlier, the most significant features driving the responses of grape (Bari et al., 2021) and olive (Boshkovski et al., 2022) genotypes to salt stress were reliably delineated by discriminant analysis.In our study, the overall classification accuracy of LDA was 80.21%, quite similar to the values reported in Boshkovski et al. (2022).
Salinity-triggered declines in P n varied between 20.47% (CSJ-18) and 83.37% (CSJ-1).Interestingly, the genotypes showing the largest salt-induced decreases in P n (CSJ-1 and CSJ-13) also had the highest P n rates in the absence of salt (Mousavi et al., 2019).The decreases in P n also seemed to be largely independent of leaf Na + and Cl − contents.For instance, despite quite similar increases in leaf Na + (109.16 and 97.89%, respectively), genotypes CSJ-18 and CSJ-42 showed remarkably different reductions in P n (i.e., 20.46 and 58.37%, respectively).Similarly, genotypes CSJ-1 and CSJ-18 showed 83.38 and 20.46% decreases in P n , respectively, despite salt-induced increases in leaf Cl − being 19.23 and 80.77%, respectively (Alipour, 2018;Hussain et al., 2012).The tested genotypes showed varying degrees of reductions in transpiration rate (E) and internal CO 2 (C i ).Despite being crucial for regulating ion absorption, reduced transpiration can impede plant growth by lowering the photosynthesis (Negrão, Schmöckel & Tester, 2017).The activity of antioxidant enzymes also showed significant genotypic differences.For instance, while APX activity increased markedly (>70.0%) in response to salt in CSJ-18 and CSJ-19, it increased by only ~28.0% in CSJ-1.CSJ-1 also displayed the lowest (~8.0%) increase in CAT activity while it was the highest (63.48%) in CSJ-18.When exposed to salt, genotypes CSJ-18 and CSJ-19 also showed significantly higher levels of POX and SOD than other genotypes.This implies that jamun genotypes react differently in terms of antioxidant enzyme activity to oxidative stress brought on by salt (Ayaz et al., 2021;Singh et al., 2023).The antioxidant enzymes shield the salt-stressed plants from oxidative damage by detoxifying the ROS and regulating ROS formation (Moradbeygi et al., 2020), and are believed to be potential markers for identifying the salt-tolerant genotypes (Sorkheh et al., 2012).We noticed that genotypes CSJ-18 and CSJ-19 were particularly efficient in upregulating anti-oxidant enzymes.Certain genotypes are frequently better at fending-off oxidative damage caused by salt because they have stronger antioxidant defenses (Abid et al., 2020;Ayaz et al., 2021;Etehadpour et al., 2020).Genotypic variations in antioxidant activities can be attributed to their intricate expression (Racchi, 2013) and organelle-specific activities (Niu & Liao, 2016) within plant cells.

CONCLUSIONS
Our results demonstrated distinct genotypic responses to salt within both mono-and poly-embryonic types.The decreases brought on by salt in leaf, stem, root and whole plant biomass were relatively greater in monoembryonic than in polyembryonic types.Furthermore, most polyembryonic genotypes exhibited lower or much lower reductions in root than in shoot mass when treated with salt, suggesting that they might be more adept at absorbing water and nutrients in saline soils.Despite significant increases in Na + and Cl -in different plant parts, leaf tip burn and marginal scorch were not seen in the majority of the genotypes.While this raised the possibility of tissue tolerance, which assists in storing excess Na + and Cl − in vacuoles, we also assume that preferential accumulation of K + , Ca 2+ and Mg 2+ may have played a role in osmotic adjustment and decreased Na + uptake.Discriminant analysis suggested that while stem and root Cl -were likely responsible for the common reactions, root Na + , leaf K + and leaf Cl − accounted for the divergent responses to salt of the mono-and poly-embryonic types.Some polyembryonic genotypes (CSJ-7, CSJ-8, CSJ-14 and CSJ-19), found to be least affected by salt treatment, could be used as salt-tolerant rootstocks.All the genotypes evaluated by us, including the promising polyembryonic types which propagate true-to-type from seeds, are being maintained in a salt-affected field for further investigation and use.Future studies should aim at delineating the plausible factors accounting for tissue tolerance to excess Na + and Cl − , and preferential uptake of K + , Ca 2+ and Mg 2+ .

Table 1
Basic details of jamun genotypes used in the study.

Table 2
Effects of salinity, genotype and their interaction on biomass traits in monoembryonic and polyembryonic types.

Table 3
Effects of salinity, genotype and their interaction on leaf mineral ions in monoembryonic and polyembryonic types.

Table 4
Effects of salinity, genotype and their interaction on stem ion contents in monoembryonic and polyembryonic types.

Table 5
Effects of salinity, genotype and their interaction on root ion contents in monoembryonic and polyembryonic types.

Table 6
Analysis of variance and mean comparisons for testing the effects of salinity (S), genotype (G) and their interaction (S Â G) on variance in gas exchange attributes and anti-oxidant enzymes.